A positive control is required. If the positive control is negative too, then it is not necessarily a true negative. You can never proove with 100% certainty that a negative PCR is negative because there is no target. There is always a chance that just this particular reaction did not amplify a present target beyond the detection limit. However, having a positive result of the positive-control, it is at least quite likely that the negative result is due to the absence of a target.

I guess a contamination model like this: use you positive control sample serially diluted with (A) UHQ water, (B) your negative sample. If the signal in series B is not lost, there are no potent inhibitors of PCR so the sample is true negative. The constraint of method is you cannot detect a false negative.

A positive control is required. If the positive control is negative too, then it is not necessarily a true negative. You can never proove with 100% certainty that a negative PCR is negative because there is no target. There is always a chance that just this particular reaction did not amplify a present target beyond the detection limit. However, having a positive result of the positive-control, it is at least quite likely that the negative result is due to the absence of a target.

I agree with the above, you could also try a multiplex reaction, with another set of primers to ensure no PCR inhibitors are present, as long as you can descriminate the respective product from the control and the test (bear in mind multiplexing often causes the odd surprise that you didn't predict). Or at least another PCR of a diferent target (that you would expect to be positive) with the same template wil add to the evidence that the negative is negative that the suggestions above aim to provide.

To be a bit more specific, you can do two positive controls along with your test PCRs. 1) use your product-specific primers to PCR your gene from a sample that you know should give product. This tells you that your reagents are working and the conditions are right. 2) use primers against a housekeeping gene (such as actin or B-catenin) that is always expressed. This will tell you that your DNA prep has worked and that your samples all contain equal amounts (or not) of DNA. Good luck!

Having positive results of positive control is an indication for successful PCR. If you sure there is no target, negative sample PCR is truly negative.

You can include more than one negative control with no target.

But i agree with the above you can never proove with 100% certainty that a negative PCR is negative because there is no target. There is always a chance that just this particular reaction did not amplify a present target (mistake) beyond the detection limit or inhibitor of PCR reaction present.

You must use a internal kontrol to confirm negative PCR product.

Hi Surya,

for clinical samples with high content of inhibitors, like plasma samples, besides the regular positive and negative controls, and the PCR control, we use to include an additional reaction (tube) with an internal control (spike), which can be a exogenous DNA or plasmid, with the same or different target.

The results interpretations is like that

Pacient Rxn: + Spike: + Positive control + -Negative Control - : Presençe of target

Pacient Rxn: - Spike: + Positive control + -Negative Control - : Absence of target (at the sensitivity of the assay)

Pacient Rxn: - Spike: + Positive control + -Negative Control - : Absence of target

Pacient Rxn: - Spike: - Positive control + -Negative Control - : Inconclusive (presence of inhibitors) REPEAT EXTRACTION

Pacient Rxn: -/+ Spike: -/+ Positive control + -Negative Control + : Contaminated reaction REPEAT EXTRACTION/REACTION

Pacient Rxn: - Spike: - Positive control + -Negative Control - : Failed reaction REPEAT REACTION

Hope to habe been useful! Best luck.

Sorry,

Failed reaction should be

Pacient Rxn: - Spike: - Positive control - Negative Control - : Failed reaction REPEAT REACTION

Always incorporate an internal amplification control for your PCR reaction. It can be either competetive or non-competetive. I personally would prefer competetive control. Please go through this article by Hoorfar. Practical Considerations in Design of Internal Amplification Controls for Diagnostic PCR Assays by Hoorfar J etal., 2004

Use positive control to overcome this crisis.